CN105998065A - Carbon nanosphere preparation as well as preparation method and anti-helicobacter pylori infection experiment method thereof - Google Patents
Carbon nanosphere preparation as well as preparation method and anti-helicobacter pylori infection experiment method thereof Download PDFInfo
- Publication number
- CN105998065A CN105998065A CN201610519621.XA CN201610519621A CN105998065A CN 105998065 A CN105998065 A CN 105998065A CN 201610519621 A CN201610519621 A CN 201610519621A CN 105998065 A CN105998065 A CN 105998065A
- Authority
- CN
- China
- Prior art keywords
- nano carbon
- carbon balls
- preparation
- balls grain
- helicobacter pylori
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/44—Elemental carbon, e.g. charcoal, carbon black
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Urology & Nephrology (AREA)
- Toxicology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Inorganic Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses a carbon nanosphere preparation as well as a preparation method and an anti-helicobacter pylori infection experiment method thereof. The carbon nanosphere preparation is prepared by mixing carbon nanospheres and dispersion liquid; the concentration of the carbon nanosphere preparation is 0.1 percent to 1.0 percent; the dispersion liquid is de-ionized water and the grain diameter of not less than 95 percent of the carbon nanospheres is 5nm to 30nm. The carbon nanosphere preparation has the advantages of low cost, easiness for manufacturing, good stability, high safety and good anti-helicobacter pylori infection effect. The preparation method of the carbon nanosphere preparation has the characteristics of easiness of implementation, simple process and convenience for operation. The anti-helicobacter pylori infection experiment method of the carbon nanosphere preparation has the characteristics of convenience for implementation, scientificity and rationality. The carbon nanosphere preparation disclosed by the invention can be developed as a new drug for treating helicobacter pylori infection, so that a new idea is provided for developing anti-helicobacter pylori infection medicines and a new strategy is provided for prevention and treatment of anti-helicobacter pylori infection.
Description
Technical field
The present invention relates to a kind of carbon nanomaterial, especially relate to a kind of Nano carbon balls grain preparation and preparation method thereof, and this carbon
The experimental technique of nanometer spherolite preparation Helicobacter pylori Infection.
Background technology
The infectious relevant disease of helicobacter pylori (Helicobacter pylori, Hp), as gastritis, gastric ulcer, duodenum are burst
The healthy of the mankind in infections and gastric cancer serious harm.According to World Health Organization (WHO) (WHO) statistics, the whole world there are about 50%
The helicobacter pylori state of an illness that population infects helicobacter pylori, especially China is the severeest, and the people of about 60% infects helicobacter pylorus
Bacterium, helicobacter pylori has been classified as a class cancerigenic factor of gastric cancer by WHO.At present, treatment helicobacter pylori infections is anti-by using
Raw element and the conjoint therapy of acid inhibitor.But owing to the drug resistance of antibiotic is increasingly severe, cause dosage to strengthen, treat
Cheng Yanchang, and increase the weight of along with adverse effect, problem that relapse rate still remains high.Therefore, clinical in the urgent need to non-
The new therapy of antibiotic and the New Policy of Helicobacter pylori Infection, new material.
Carbon nanomaterial is the nanotechnology material that a class is novel, has multiple use.CNT in carbon nanomaterial has
Antibacterial action, and the Nano carbon balls grain (Carbon nanospheres, CNS) in carbon nanomaterial has transporting drugs and fluorescence
Spikes etc. act on.Owing to Nano carbon balls grain has structure and the characteristic of uniqueness, its potential biomedical applications has to be developed.Mesh
Before, also there is no the research report of Nano carbon balls grain antibacterial action, there is no the research report of Nano carbon balls grain anti-helicobacter pylori yet.
The helicobacter pylori harm to body, is on the one hand the effect of helicobacter pylori itself, is on the other hand its metabolite such as carbamide
The effect of enzyme.Helicobacter pylori can produce a large amount of urease, and urease plays the effect of field planting and virulence in helicobacter pylori infections,
Being virulence factor, urease is also the target spot for the treatment of helicobacter pylori simultaneously.Therefore, if Nano carbon balls grain or its preparation can
Directly suppress or kill helicobacter pylori thalline itself, can suppress or destroy again the urease of helicobacter pylori, then this carbon is received
Rice spherolite and preparation thereof just have huge anti-helicobacter pylori application prospect.
Although the nano-carbon powder in carbon nanomaterial being made the research of Eradication Therapy of Helicobacter pylori Infection agent, such as Application No.
The patent application of 2014104902353 and 2014104898659, but the nano-carbon powder diameter of particle more than 95% that both is selected
Less than or equal to 5nm；Electronegative, its therapeutic agent uses the dispersion preparation of human body isosmotic solution, and its pH value is controlled in 7.8～9.1.
Its problem being primarily present following several respects: 1) owing to its more than 95% nano-carbon powder diameter of particle is for less than or equal to 5nm, because of it
Particle diameter is the least penetrates readily through entrance human body cell, it is easier to produces toxicity or strengthens toxicity；2) because isosmotic solution is containing a large amount of cationes,
Positively charged, it can produce certain impact to the long-time stability of the nano-carbon powder powder body rich in negative charge.
Summary of the invention
It is an object of the invention to provide a kind of Nano carbon balls grain preparation and preparation method thereof and Helicobacter pylori Infection experimental technique,
Wherein, Nano carbon balls grain preparation have with low cost, preparation easily, the high advantage of good stability, safety, preparation method has
Have implement easily, feature that technique is simple and convenient to operate, experimental technique has implements convenient, scientific and reasonable feature.
It is increasingly severe for solving prior art is treated its antibiotic resistance existed of traditional remedies of helicobacter pylori infections,
Cause dosage to strengthen, the course for the treatment of extends, adverse effect increases the weight of the problem high with infection and recurrence rate, and the present invention carries
A kind of Nano carbon balls grain preparation of confession, is mixed by Nano carbon balls grain and dispersion liquid and constitutes, and the concentration of Nano carbon balls grain preparation is
0.1%～1.0%, wherein, dispersion liquid is deionized water, and the particle diameter of the Nano carbon balls grain of more than 95% is 5～30nm.
Further, one Nano carbon balls grain preparation of the present invention, wherein, the pH value of described Nano carbon balls grain preparation is 7.1～7.6.
Further, one Nano carbon balls grain preparation of the present invention, wherein, the particle diameter frequency percentage ratio of described Nano carbon balls grain is:
Particle diameter be 5～10nm Nano carbon balls grain account for 20～30%, particle diameter be 10～15nm Nano carbon balls grain account for 25～35%, grain
Footpath be 15～20nm Nano carbon balls grain account for 15～25%, particle diameter be 20～25nm Nano carbon balls grain account for 6～10%, grain
Footpath be 25～30nm Nano carbon balls grain account for 1～7%.
The preparation method of a kind of Nano carbon balls grain preparation that the present invention provides, comprises the following steps:
One, Nano carbon balls grain is prepared with Graphene for original material processing；
Two, utilizing transmission electron microscope to screen particle diameter satisfactory Nano carbon balls grain, wherein, particle diameter is the Nano carbon balls of 5～30nm
Grain accounts for more than 95%；Nano carbon balls grain is single spherical dispersion；
Three, according to concentration requirement, weigh the Nano carbon balls grain after step 2 is screened respectively and measure deionized water, standby；
Four, Nano carbon balls grain step 3 obtained and deionized water mixing dispersion；
Five, being stirred the mixed liquor being mixed to get through step 4, wherein, mixing time is 10～20 minutes, stirring
Speed is 60～80r/min；
Six, the mixed liquor after step 5 stir process carrying out supersound process, wherein, ultrasonic time is 15～20 minutes, super
Sound intensity is 80～100w；
Seven, utilize sodium bicarbonate sodium carbonate buffer that the mixed liquor after step 6 supersound process is adjusted pH value to process,
The pH value of mixed liquor is adjusted to 7.1～7.6, obtains final Nano carbon balls grain preparation.
Further, the preparation method of the present invention a kind of Nano carbon balls grain preparation, wherein, particle diameter described in step 2 be 5～
The Nano carbon balls grain of 30nm accounts for more than 95%, and wherein, the particle diameter frequency percentage ratio of Nano carbon balls grain is: particle diameter is 5～10nm
Nano carbon balls grain account for 20～30%, particle diameter be 10～15nm Nano carbon balls grain account for 25～35%, particle diameter is 15～20nm
Nano carbon balls grain account for 15～25%, particle diameter be 20～25nm Nano carbon balls grain account for 6～10%, particle diameter is 25～30nm
Nano carbon balls grain accounts for 1～7%.
Further, the preparation method of the present invention a kind of Nano carbon balls grain preparation, wherein, to through step 5 described in step 6
Mixed liquor after stir process carries out supersound process, carries out supersound process under low temperature water-bath.
Further, the preparation method of the present invention a kind of Nano carbon balls grain preparation, wherein, at ultrasonic time described in step 6 be
15～20 minutes, referring to that ultrasonic all times is 15～20 minutes, wherein, single ultrasonic time is 3 seconds, and off time is 3
Second.
The experimental technique of a kind of Nano carbon balls grain preparation Helicobacter pylori Infection that the present invention provides, comprises the following steps:
I, by the external RUT experiment detection Nano carbon balls grain preparation suppression ratio to helicobacter Pylori urease；
II, the coup injury effect to helicobacter pylori of the transmission electron microscope observing Nano carbon balls grain preparation is utilized；
III, the characteristic of Nano carbon balls grain preparation Helicobacter pylori Infection is determined according to the interpretation of result of step I and step II.
Further, the experimental technique of the present invention a kind of Nano carbon balls grain preparation Helicobacter pylori Infection, wherein, in step I
Described in by external RUT experiment detection the Nano carbon balls grain preparation suppression ratio to helicobacter Pylori urease, including with
Lower step:
A, preparation and diluted sample: with normal saline by Helicobacter pylori SS1 bacterium solution 109CFU/ml dilutes 1000 times
To 106CFU/ml, standby；By three kinds of Nano carbon balls grain preparation deionized waters of concentration respectively 1.0%, 0.5%, 0.4%
Diluting 100 times, standby, the concentration of three kinds of Nano carbon balls grain preparations after dilution is respectively 100ug/ml, 50ug/ml, 40ug/ml；
Urease inhibitor acetohrdroxamic acid is first dissolved into 1% with deionized water, dilutes 100 times with deionized water the most again, standby；
Urease inhibitor acetohrdroxamic acid concentration after dilution is 100ug/ml；Remove ionized water, standby；Wherein, Urease inhibitor
Agent acetohrdroxamic acid is as positive control, and deionized water is as negative control；
B, loading: in the hole of 96 orifice plates, add the 10 of 10ul6CFU/ml bacterium solution, multiple three holes；After adding, according to packet point
It is not rapidly added tri-kinds of Nano carbon balls grain preparations of 100ug/ml, 50ug/ml, 40ug/ml, deionized water, 100ug/ml acetyl oxygen
The oxime each 90ul of acid；
C, hatch: use sealed membrane to seal 96 orifice plates after loading, and in the calorstat of 4 DEG C, hatch 24 hours；
D, colour developing: after having hatched, add the urea reagent of 50ul in corresponding hole, and be put in the calorstat of 37 DEG C and hatch
30 points of kind colour developings；
E, reading: after hatching 30 points of kinds, reading at 578nm wavelength, and record the OD value often organized；
F, calculating suppression ratio: suppression ratio=[1-(ODMeasure-ODBlank)/(ODComparison-ODBlank)] × 100%.
Further, the experimental technique of the present invention a kind of Nano carbon balls grain preparation Helicobacter pylori Infection, wherein, in step II
Described in utilize the coup injury effect to helicobacter pylori of the transmission electron microscope observing Nano carbon balls grain preparation, comprise the following steps:
Prepared by g, bacterium solution: with normal saline by helicobacter pylori NTCC11637 bacterium solution 109CFU/ml dilutes 10 times extremely
108CFU/ml, standby；
H, loading: take 12 1ml test tubes, in often pipe, add the 10 of 360ul8CFU/ml helicobacter pylori NTCC11637
Bacterium solution, multiple three holes；After adding, according to packet be rapidly added respectively 0.4%, 0.1% Nano carbon balls grain preparation, deionized water,
The 0.1% each 40ul of clarithromycin, mixing；Wherein, deionized water is as negative control, and clarithromycin is as positive control；
I, reaction: using sealed membrane to seal orifice plate after loading, first the constant-temperature table at 37 DEG C shakes bacterium and cultivates 10 points of kinds, then takes
Go out and put into rapidly and the calorstat of 37 DEG C is cultivated 30 points of kinds；
J, dyeing: take 100ul reactant liquor and be placed on transmission electron microscope copper mesh, and with 1.0% phosphotungstic acid negative staining；
K, observation: under transmission electron microscope, observe the Morphological Features of helicobacter pylori, and judge that Nano carbon balls grain preparation is to helicobacter pylorus
Bacterium coup injury result.
One Nano carbon balls grain preparation of the present invention and preparation method thereof and Helicobacter pylori Infection experimental technique and prior art phase
Ratio, has the advantage that the Nano carbon balls grain preparation that (1) present invention provides uses the Nano carbon balls grain of non-antibiotic to be that preparation is former
Material, and by more than 95% spherolite size controlling 5～30nm, it is to avoid because particle diameter is too small, body is produced toxicity or increase
The problem of toxicity, it is provided that safety.(2) use deionized water as dispersion liquid, make full use of Nano carbon balls grain surface many
Hole, enhances water absorption, makes Nano carbon balls grain dispersion rate in deionized water reach more than 99.5%, and because deionized water does not contains
Other metal cations, make electronegative Nano carbon balls grain not affected by by cation, disperse in single spherical, keep away in water
Exempt from the problem that spherolite is reunited, it is ensured that the high stability of Nano carbon balls grain preparation.(3) present invention is by by Nano carbon balls grain
The pH value of preparation controls 7.1～7.6, reduces or eliminates in Nano carbon balls grain preparation PH (acid-resisting) to helicobacter pylori
The impact of growth, more can highlight the embodiment Nano carbon balls grain antibacterial action to helicobacter pylori itself.(4) the carbon nanometer of the present invention
Spherolite preparation have with low cost, preparation easily, the high advantage of good stability, safety, its preparation method have enforcement easily,
The advantage that technique is simple and convenient to operate, experimental technique has implements convenient, scientific and reasonable advantage.(5) experiment shows, this
The Nano carbon balls grain preparation of invention has prominent and powerful anti-helicobacter pylori effect, suppresses helicobacter Pylori urease activity
Rate is high, and required dosage is little；Nano carbon balls grain has coup injury effect to helicobacter pylori, to helicobacter pylori flagellum and cell membrane
Cell wall damage is obvious.(6) the Nano carbon balls grain preparation of the present invention can as treatment helicobacter pylori infections new drug development, for
The medicine of exploitation anti-helicobacter pylori opens new thinking, also provides new strategy by the preventing and treating for Helicobacter pylori Infection.
The most shown detailed description of the invention is to one Nano carbon balls grain preparation of the present invention and preparation method thereof and anti-pylorus spiral shell
Bacillus infection experimental technique is described in further detail:
Accompanying drawing explanation
Fig. 1 is to observe in the coup injury experiment to helicobacter pylori of the Nano carbon balls grain preparation by transmission electron microscope (TEM),
The helicobacter pylori aspect graph of 400ug/ml high dose Nano carbon balls grain preparation group；
Fig. 2 is to observe in the coup injury experiment to helicobacter pylori of the Nano carbon balls grain preparation by transmission electron microscope (TEM),
The helicobacter pylori aspect graph of 100ug/ml low dosage Nano carbon balls grain preparation group；
Fig. 3 is to observe in the coup injury experiment to helicobacter pylori of the Nano carbon balls grain preparation by transmission electron microscope (TEM),
The helicobacter pylori aspect graph of 100ug/ml clarithromycin group；
Fig. 4 is to be observed in the coup injury experiment to helicobacter pylori of the Nano carbon balls grain preparation by transmission electron microscope (TEM), goes
The helicobacter pylori aspect graph of ionized water group.
Detailed description of the invention
Firstly the need of explanation, this invention provides both a kind of Nano carbon balls grain preparation, provide again its preparation method, simultaneously
Additionally provide the experimental technique of Nano carbon balls grain preparation Helicobacter pylori Infection.
As detailed description of the invention, Nano carbon balls grain preparation is made up of Nano carbon balls grain and dispersion liquid mixing, and makes its concentration control
0.1%～1.0%.Wherein, dispersion liquid is deionized water, and the particle diameter of the Nano carbon balls grain of more than 95% is 5～30nm.For subtracting
Little or eliminate the PH (acid-resisting) impact on growth of H. pylori in Nano carbon balls grain preparation, more can highlight embodiment carbon nanometer
The spherolite antibacterial action to helicobacter pylori itself, this detailed description of the invention allows the pH value of Nano carbon balls grain preparation be 7.1～7.6.
As prioritization scheme, this detailed description of the invention makes the particle diameter frequency percentage ratio of Nano carbon balls grain use values below: allow particle diameter
Be 5～10nm Nano carbon balls grain account for 20～30%, the Nano carbon balls grain allowing particle diameter be 10～15nm accounts for 25～35%, allows grain
Footpath be 15～20nm Nano carbon balls grain account for 15～25%, the Nano carbon balls grain allowing particle diameter be 20～25nm accounts for 6～10%, allows
Particle diameter be 25～30nm Nano carbon balls grain account for 1～7%.
The Nano carbon balls grain preparation that the present invention provides uses the Nano carbon balls grain of non-antibiotic to be preparation raw material, and by more than 95%
Spherolite size controlling 5～30nm, it is to avoid because particle diameter is too small body produced toxicity or the problem increasing toxicity, it is provided that
Safety.And by using deionized water to enhance water absorption as dispersion liquid, the porous making full use of Nano carbon balls grain surface,
Make Nano carbon balls grain dispersion rate in deionized water reach more than 99.5%, and because deionized water is without other metal cations, make
Electronegative Nano carbon balls grain do not affected by by cation, disperses in single spherical in water, it is to avoid the problem that spherolite is reunited,
Ensure that the high stability of Nano carbon balls grain preparation.Meanwhile, the present invention provide Nano carbon balls grain preparation have with low cost,
Preparation easily, the high advantage of good stability, safety.Experiment shows, the Nano carbon balls grain preparation that the present invention provides has prominent
And powerful anti-helicobacter pylori effect, high to helicobacter Pylori urease maximum inhibition, required dosage is little.Nano carbon balls grain
Helicobacter pylori is had coup injury effect, obvious to helicobacter pylori flagellum and cell membrane cell wall damage.The carbon of the present invention is received
Rice spherolite preparation can be as the new drug development for the treatment of helicobacter pylori infections, and the medicine for exploitation anti-helicobacter pylori opens new
Thinking, also provides new strategy by the preventing and treating for Helicobacter pylori Infection.
As detailed description of the invention, the preparation method of Nano carbon balls grain preparation, comprise the following steps:
One, Nano carbon balls grain is prepared with Graphene for original material processing；
Two, utilizing transmission electron microscope to screen particle diameter satisfactory Nano carbon balls grain, wherein, particle diameter is the Nano carbon balls of 5～30nm
Grain accounts for more than 95%；Nano carbon balls grain is single spherical dispersion；
Three, according to concentration requirement, weigh the Nano carbon balls grain after step 2 is screened respectively and measure deionized water, standby；
Four, Nano carbon balls grain step 3 obtained and deionized water mixing dispersion；
Five, being stirred the mixed liquor being mixed to get through step 4, wherein, mixing time is 10～20 minutes, stirring
Speed is 60～80r/min；
Six, the mixed liquor after step 5 stir process carrying out supersound process, wherein, ultrasonic time is 15～20 minutes, super
Sound intensity is 80～100w；
Seven, sodium bicarbonate sodium carbonate buffer is utilized to carry out the mixed liquor after step 6 supersound process adjusting pH value to process,
The pH value of mixed liquor is adjusted to 7.1～7.6, obtains final Nano carbon balls grain preparation.
In above-mentioned steps two, described particle diameter be 5～30nm Nano carbon balls grain account for more than 95%, wherein, Nano carbon balls grain
Particle diameter frequency percentage ratio is: particle diameter be 5～10nm Nano carbon balls grain account for 20～30%, particle diameter is the carbon nanometer of 10～15nm
Spherolite accounts for 25～35%, particle diameter be 15～20nm Nano carbon balls grain account for 15～25%, particle diameter is the carbon nanometer of 20～25nm
Spherolite accounts for 6～10%, particle diameter be 25～30nm Nano carbon balls grain account for 1～7%.
In above-mentioned steps six, described mixed liquor after step 5 stir process is carried out supersound process, be under low temperature water-bath
Carry out supersound process.
In above-mentioned steps six, described ultrasonic time is 15～20 minutes, refers to that ultrasonic all times is 15～20 minutes, its
In, single ultrasonic time is 3 seconds, and off time is 3 seconds.
Embodiment 1: prepare Nano carbon balls grain and deionized water.
Prepare Nano carbon balls grain with Graphene for original material processing, under transmission electron microscope, screen the carbon nanometer that particle diameter is 5～30nm
Spherolite, standby.Should ensure that the Nano carbon balls grain that particle diameter is 5～30nm accounts for more than the 95% of total amount.
Prepare deionized water, standby.
Embodiment 2: the Nano carbon balls grain preparation of preparation 1.0%.
According to the concentration requirement of 1.0%, take quantitative Nano carbon balls grain and deionized water mixing dispersion, and stir 20 minutes, its
Middle mixing speed is 80r/min.
Mixed liquor after stirring is carried out supersound process, and the most ultrasonic all times is set to 20 minutes, and single ultrasonic time is set to 3
Second, it being set to 3 seconds off time, ultrasound intensity is set to 100w (ultrasonic under low temperature water-bath).
Sodium bicarbonate sodium carbonate buffer is used to carry out adjusting pH value to process the mixed liquor after ultrasonic, and final by mix
PH value is adjusted to 7.6, to obtain satisfactory Nano carbon balls grain preparation.
Embodiment 3: the Nano carbon balls grain preparation of preparation 0.5%.
According to the concentration requirement of 0.5%, take quantitative Nano carbon balls grain and deionized water mixing dispersion, and stir 15 minutes, its
Middle mixing speed is 70r/min.
Mixed liquor after stirring is carried out supersound process, and the most ultrasonic all times is set to 18 minutes, and single ultrasonic time is set to 3
Second, it being set to 3 seconds off time, ultrasound intensity is set to 100w (ultrasonic under low temperature water-bath).
Sodium bicarbonate sodium carbonate buffer is used to carry out adjusting pH value to process the mixed liquor after ultrasonic, and final by mix
PH value is adjusted to 7.4, to obtain satisfactory Nano carbon balls grain preparation.
Embodiment 4: the Nano carbon balls grain preparation of preparation 0.2%.
According to the concentration requirement of 0.2%, take quantitative Nano carbon balls grain and deionized water mixing dispersion, and stir 10 minutes, its
Middle mixing speed is 60r/min.
Mixed liquor after stirring is carried out supersound process, and the most ultrasonic all times is set to 15 minutes, and single ultrasonic time is set to 3
Second, it being set to 3 seconds off time, ultrasound intensity is set to 80w (ultrasonic under low temperature water-bath).
Sodium bicarbonate sodium carbonate buffer is used to carry out adjusting pH value to process the mixed liquor after ultrasonic, and final by mix
PH value is adjusted to 7.2, to obtain satisfactory Nano carbon balls grain preparation.
Embodiment 5: the Nano carbon balls grain preparation of preparation 0.1%.
According to the concentration requirement of 0.2%, take quantitative Nano carbon balls grain and deionized water mixing dispersion, and stir 10 minutes, its
Middle mixing speed is 60r/min.
Mixed liquor after stirring is carried out supersound process, and the most ultrasonic all times is set to 15 minutes, and single ultrasonic time is set to 3
Second, it being set to 3 seconds off time, ultrasound intensity is set to 80w (ultrasonic under low temperature water-bath).
Sodium bicarbonate sodium carbonate buffer is used to carry out adjusting pH value to process the mixed liquor after ultrasonic, and final by mix
PH value is adjusted to 7.1, to obtain satisfactory Nano carbon balls grain preparation.
As the detailed description of the invention of the experimental technique of Nano carbon balls grain preparation Helicobacter pylori Infection, comprise the following steps:
I, by the external RUT experiment detection Nano carbon balls grain preparation suppression ratio to helicobacter Pylori urease；
II, the coup injury effect to helicobacter pylori of the transmission electron microscope observing Nano carbon balls grain preparation is utilized；
III, the final result of Nano carbon balls grain preparation Helicobacter pylori Infection is determined according to the interpretation of result of step I and step II.
Embodiment 6: by the external RUT experiment detection Nano carbon balls grain preparation suppression ratio to helicobacter Pylori urease.
Preparation and diluted sample: with normal saline by Helicobacter pylori SS1 bacterium solution 109CFU/ml (OD578=0.6)
Dilute 1000 times to 106CFU/ml, standby；Concentration is respectively three kinds of Nano carbon balls grain preparations of 1.0%, 0.5%, 0.4%
With deionized water dilute 100 times, standby, the concentration of three kinds of Nano carbon balls grain preparations after dilution be respectively 100ug/ml, 50ug/ml,
40ug/ml (being that PH is neutral with deionized water sample after the dilution of 100 times)；Urease inhibitor acetohrdroxamic acid is first used
Deionized water dissolving becomes 1.0%, dilutes 100 times with deionized water the most again, standby；Urease inhibitor acetyl oxygen after dilution
Oxime acid concentration is 100ug/ml；Remove ionized water, standby；Wherein, urease inhibitor acetohrdroxamic acid as positive control,
Deionized water is as negative control.
Loading: add the 10 of 10ul in the hole of 96 orifice plates6CFU/ml bacterium solution, multiple three holes；After adding, according to packet respectively
It is rapidly added tri-kinds of Nano carbon balls grain preparations of 100ug/ml, 50ug/ml, 40ug/ml, deionized water, 100ug/ml acetyl oxygen oxime
The each 90ul of acid.
Hatch: use sealed membrane to seal 96 orifice plates after loading, and in the calorstat of 4 DEG C, hatch 24 hours.
Colour developing: after having hatched, adds the urea reagent of 50ul in corresponding hole, and is put in the calorstat of 37 DEG C and hatches 30
Divide kind of a colour developing.
Reading: after hatching 30 points of kinds, reading at 578nm wavelength, and record the OD value often organized.
Calculating suppression ratio: suppression ratio=[1-(ODMeasure-ODBlank)/(ODComparison-ODBlank)] × 100%.
OD value and suppression ratio result such as table 1
Table 1: Nano carbon balls grain agent in vitro RUT inhibitory enzyme activity experimental data table
By experimental result listed by table 1, the Nano carbon balls grain preparation of the Helicobacter pylori Infection of the present invention is to helicobacter pylorus
Bacterium urease has obvious inhibitory action.
Embodiment 7: observe the coup injury effect to helicobacter pylori of the Nano carbon balls grain preparation by transmission electron microscope (TEM).
Prepared by bacterium solution: with normal saline by helicobacter pylori NTCC11637 bacterium solution 109CFU/ml (OD578=0.6) dilutes 10
Again to 108CFU/ml, standby.
Loading: take 12 1ml test tubes, adds 360ul 10 in often pipe8The helicobacter pylori NTCC11637 bacterium of CFU/ml
Liquid, multiple three holes；After adding, according to packet be rapidly added respectively 0.4%, 0.1% Nano carbon balls grain preparation, deionized water,
The 0.1% each 40ul of clarithromycin, and mix；Wherein, deionized water is as negative control, and clarithromycin is as positive control.
Reaction: using sealed membrane to seal orifice plate after loading, first the constant-temperature table at 37 DEG C shakes bacterium and cultivates 10 points of kinds, then takes out
And put into rapidly 30 points of kinds of cultivation in the calorstat of 37 DEG C.
Dyeing: take 100ul reactant liquor and be placed on transmission electron microscope copper mesh, and with 1.0% phosphotungstic acid negative staining.
Observe: under transmission electron microscope (TEM), observe the Morphological Features of helicobacter pylori, and judge that Nano carbon balls grain preparation is to deep and remote
Helicobacter pylori coup injury result.
Under transmission electron microscope (TEM), respectively group experimental result is as shown in Figures 1 to 4:
Shown in 400ug/ml high dose Nano carbon balls grain preparation group Fig. 1: helicobacter pylori has no that flagellum, cell wall after birth are thinning the completeest
Whole, antibacterial soft edge, structure is unclear.
Shown in 100ug/ml low dosage Nano carbon balls grain preparation group Fig. 2: helicobacter pylori flagellum reduces, and cell wall after birth is imperfect.
Shown in 100ug/ml clarithromycin group Fig. 3: helicobacter pylori cell wall after birth is thinning imperfect, flagellum reduces or disappears.
Shown in deionized water group Fig. 4: helicobacter pylori flagellum enriches, cell wall after birth is complete, and antibacterial profile understands, thalline is normal.
It follows that Nano carbon balls grain formulation C NS has coup injury effect to helicobacter pylori.
Above example is only the description carrying out the preferred embodiment for the present invention, and the present invention is not claimed what scope was carried out
Limiting, on the premise of designing spirit without departing from the present invention, this area engineers and technicians make according to technical scheme
Various forms of deformation, all should fall in the protection domain that claims of the present invention determines.
Claims (10)
1. a Nano carbon balls grain preparation, it is characterised in that described Nano carbon balls grain preparation is mixed by Nano carbon balls grain and dispersion liquid
Constituting, the concentration of Nano carbon balls grain preparation is 0.1%～1.0%, and wherein, dispersion liquid is deionized water, the carbon nanometer of more than 95%
The particle diameter of spherolite is 5～30nm.
2. according to a kind of Nano carbon balls grain preparation described in claim 1, it is characterised in that described Nano carbon balls grain preparation
PH value is 7.1～7.6.
3. according to a kind of Nano carbon balls grain preparation described in claim 2, it is characterised in that the particle diameter frequency of described Nano carbon balls grain
Hundreds of proportions by subtraction are: particle diameter be 5～10nm Nano carbon balls grain account for 20～30%, particle diameter be 10～15nm Nano carbon balls grain account for
25～35%, particle diameter be 15～20nm Nano carbon balls grain account for 15～25%, particle diameter be 20～25nm Nano carbon balls grain account for
6～10%, particle diameter be 25～30nm Nano carbon balls grain account for 1～7%.
4. the preparation method of Nano carbon balls grain preparation described in a claim 2, it is characterised in that comprise the following steps:
One, Nano carbon balls grain is prepared with Graphene for original material processing；
Two, utilizing transmission electron microscope to screen particle diameter satisfactory Nano carbon balls grain, wherein, particle diameter is the Nano carbon balls of 5～30nm
Grain accounts for more than 95%；Nano carbon balls grain is single spherical dispersion；
Three, according to concentration requirement, weigh the Nano carbon balls grain after step 2 is screened respectively and measure deionized water, standby；
Four, Nano carbon balls grain step 3 obtained and deionized water mixing dispersion；
Five, being stirred the mixed liquor being mixed to get through step 4, wherein, mixing time is 10～20 minutes, stirring
Speed is 60～80r/min；
Six, the mixed liquor after step 5 stir process carrying out supersound process, wherein, ultrasonic time is 15～20 minutes, super
Sound intensity is 80～100w；
Seven, utilize sodium bicarbonate sodium carbonate buffer that the mixed liquor after step 6 supersound process is adjusted pH value to process,
The pH value of mixed liquor is adjusted to 7.1～7.6, obtains final Nano carbon balls grain preparation.
5. according to the preparation method of a kind of Nano carbon balls grain preparation described in claim 4, it is characterised in that institute in step 2
Stating the Nano carbon balls grain that particle diameter is 5～30nm and account for more than 95%, wherein, the particle diameter frequency percentage ratio of Nano carbon balls grain is: particle diameter
Be 5～10nm Nano carbon balls grain account for 20～30%, particle diameter be 10～15nm Nano carbon balls grain account for 25～35%, particle diameter is
The Nano carbon balls grain of 15～20nm accounts for 15～25%, particle diameter be 20～25nm Nano carbon balls grain account for 6～10%, particle diameter be 25～
The Nano carbon balls grain of 30nm accounts for 1～7%.
6. according to the preparation method of a kind of Nano carbon balls grain preparation described in claim 4, it is characterised in that institute in step 6
State and the mixed liquor after step 5 stir process is carried out supersound process, under low temperature water-bath, carry out supersound process.
7. according to the preparation method of a kind of Nano carbon balls grain preparation described in claim 4, it is characterised in that institute in step 6
Stating ultrasonic time is 15～20 minutes, refers to that ultrasonic all times is 15～20 minutes, and wherein, single ultrasonic time is 3 seconds,
Off time is 3 seconds.
8. the experimental technique of Nano carbon balls grain preparation Helicobacter pylori Infection described in a claim 3, it is characterised in that bag
Include following steps:
I, by the external RUT experiment detection Nano carbon balls grain preparation suppression ratio to helicobacter Pylori urease；
II, the coup injury effect to helicobacter pylori of the transmission electron microscope observing Nano carbon balls grain preparation is utilized；
III, the characteristic of Nano carbon balls grain preparation Helicobacter pylori Infection is determined according to the interpretation of result of step I and step II.
9., according to the experimental technique of a kind of Nano carbon balls grain preparation Helicobacter pylori Infection described in claim 8, its feature exists
In, by external RUT experiment detection Nano carbon balls grain preparation, helicobacter Pylori urease is pressed down described in step I
Rate processed, comprises the following steps:
A, preparation and diluted sample: with normal saline by Helicobacter pylori SS1 bacterium solution 109CFU/ml dilutes 1000 times
To 106CFU/ml, standby；By three kinds of Nano carbon balls grain preparation deionized waters of concentration respectively 1.0%, 0.5%, 0.4%
Diluting 100 times, standby, the concentration of three kinds of Nano carbon balls grain preparations after dilution is respectively 100ug/ml, 50ug/ml, 40ug/ml；
Urease inhibitor acetohrdroxamic acid is first dissolved into 1% with deionized water, dilutes 100 times with deionized water the most again, standby；
Urease inhibitor acetohrdroxamic acid concentration after dilution is 100ug/ml；Remove ionized water, standby；Wherein, Urease inhibitor
Agent acetohrdroxamic acid is as positive control, and deionized water is as negative control；
B, loading: in the hole of 96 orifice plates, add the 10 of 10ul6CFU/ml bacterium solution, multiple three holes；After adding, according to packet point
It is not rapidly added tri-kinds of Nano carbon balls grain preparations of 100ug/ml, 50ug/ml, 40ug/ml, deionized water, 100ug/ml acetyl oxygen
The oxime each 90ul of acid；
C, hatch: use sealed membrane to seal 96 orifice plates after loading, and in the calorstat of 4 DEG C, hatch 24 hours；
D, colour developing: after having hatched, add the urea reagent of 50ul in corresponding hole, and be put in the calorstat of 37 DEG C and hatch
30 points of kind colour developings；
E, reading: after hatching 30 points of kinds, reading at 578nm wavelength, and record the OD value often organized；
F, calculating suppression ratio: suppression ratio=[1-(ODMeasure-ODBlank)/(ODComparison-ODBlank)] × 100%.
10. according to the experimental technique of a kind of Nano carbon balls grain preparation Helicobacter pylori Infection described in claim 8, its feature
It is, described in step II, utilizes the coup injury effect to helicobacter pylori of the transmission electron microscope observing Nano carbon balls grain preparation, bag
Include following steps:
Prepared by g, bacterium solution: with normal saline by helicobacter pylori NTCC11637 bacterium solution 109CFU/ml dilutes 10 times extremely
108CFU/ml, standby；
H, loading: take 12 1ml test tubes, in often pipe, add the 10 of 360ul8CFU/ml helicobacter pylori NTCC11637
Bacterium solution, multiple three holes；After adding, according to packet be rapidly added respectively 0.4%, 0.1% Nano carbon balls grain preparation, deionized water,
The 0.1% each 40ul of clarithromycin, mixing；Wherein, deionized water is as negative control, and clarithromycin is as positive control；
I, reaction: using sealed membrane to seal orifice plate after loading, first the constant-temperature table at 37 DEG C shakes bacterium and cultivates 10 points of kinds, then takes
Go out and put into rapidly and the calorstat of 37 DEG C is cultivated 30 points of kinds；
J, dyeing: take 100ul reactant liquor and be placed on transmission electron microscope copper mesh, and with 1.0% phosphotungstic acid negative staining；
K, observation: under transmission electron microscope, observe the Morphological Features of helicobacter pylori, and judge that Nano carbon balls grain preparation is to helicobacter pylorus
Bacterium coup injury result.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610519621.XA CN105998065B (en) | 2016-06-30 | 2016-06-30 | A kind of Nano carbon balls grain preparation and preparation method thereof and Helicobacter pylori Infection experimental method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610519621.XA CN105998065B (en) | 2016-06-30 | 2016-06-30 | A kind of Nano carbon balls grain preparation and preparation method thereof and Helicobacter pylori Infection experimental method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105998065A true CN105998065A (en) | 2016-10-12 |
| CN105998065B CN105998065B (en) | 2019-01-15 |
Family
ID=57106353
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610519621.XA Expired - Fee Related CN105998065B (en) | 2016-06-30 | 2016-06-30 | A kind of Nano carbon balls grain preparation and preparation method thereof and Helicobacter pylori Infection experimental method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105998065B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110167565A (en) * | 2017-02-14 | 2019-08-23 | Gi供应公司 | Tissue stain and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103316044A (en) * | 2013-06-07 | 2013-09-25 | 逢甲大学 | Composition structure used for inhibiting gastrointestinal bacterial infections |
| CN103316043A (en) * | 2013-06-07 | 2013-09-25 | 逢甲大学 | Medical application of composition comprising carbon material and active particles |
| CN104224838A (en) * | 2014-09-23 | 2014-12-24 | 贵州特力达纳米碳素科技有限公司 | Nanometer carbon powder and application thereof |
-
2016
- 2016-06-30 CN CN201610519621.XA patent/CN105998065B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103316044A (en) * | 2013-06-07 | 2013-09-25 | 逢甲大学 | Composition structure used for inhibiting gastrointestinal bacterial infections |
| CN103316043A (en) * | 2013-06-07 | 2013-09-25 | 逢甲大学 | Medical application of composition comprising carbon material and active particles |
| CN104224838A (en) * | 2014-09-23 | 2014-12-24 | 贵州特力达纳米碳素科技有限公司 | Nanometer carbon powder and application thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110167565A (en) * | 2017-02-14 | 2019-08-23 | Gi供应公司 | Tissue stain and application thereof |
| US11452783B2 (en) | 2017-02-14 | 2022-09-27 | Gi Supply | Tissue stain and use thereof |
| CN110167565B (en) * | 2017-02-14 | 2023-04-04 | Gi供应公司 | Tissue dyes and uses thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105998065B (en) | 2019-01-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ogunyemi et al. | The bio-synthesis of three metal oxide nanoparticles (ZnO, MnO2, and MgO) and their antibacterial activity against the bacterial leaf blight pathogen | |
| Ogunyemi et al. | Biosynthesis and characterization of magnesium oxide and manganese dioxide nanoparticles using Matricaria chamomilla L. extract and its inhibitory effect on Acidovorax oryzae strain RS-2 | |
| Kelkawi et al. | Green synthesis of silver nanoparticles using Mentha pulegium and investigation of their antibacterial, antifungal and anticancer activity | |
| CN106727432B (en) | A kind of Prussian blue analogue nano particle and the preparation method and application thereof | |
| Dong et al. | Preparation of magnetically separable N-halamine nanocomposites for the improved antibacterial application | |
| CN105948124A (en) | Nano-molybdenum sulfide antibacterial material and synthetic method thereof and application thereof | |
| CN1953664A (en) | Anti-microbial activity of biologically stabilized silver nano particles | |
| Wang et al. | Inactivation of sulfonamide antibiotic resistant bacteria and control of intracellular antibiotic resistance transmission risk by sulfide-modified nanoscale zero-valent iron | |
| CN101506371A (en) | Method for producing metal nanoparticles | |
| Grande et al. | Antimicrobial and antibiofilm activities of new synthesized silver ultra-nanoclusters (SUNCs) against Helicobacter pylori | |
| CN102978241A (en) | Method for producing metal nanoparticles | |
| Karthik et al. | Influence of ball milling on the particle size and antimicrobial properties of Tridax procumbens leaf nanoparticles | |
| CN110496219A (en) | A kind of synthetic method of novel ferrihydrite nanometric photosensitizer and its application in anticancer antibiotic | |
| CN106342822A (en) | Fludioxonil, prothioconazole and chlorantraniliprole containing composition and seed coating agent and application of seed coating agent | |
| CN105998065A (en) | Carbon nanosphere preparation as well as preparation method and anti-helicobacter pylori infection experiment method thereof | |
| Pant et al. | Bi 2 O 3 nanoparticles exhibit potent broad-spectrum antimicrobial activity and the ability to overcome Ag-, ciprofloxacin-and meropenem-resistance in P. aeruginosa: the next silver bullet of metal antimicrobials? | |
| CN110655905A (en) | Multifunctional synergistic negative ion release material and preparation method thereof | |
| Najeeb et al. | Green chemistry: evolution in architecting Schemes for perfecting the synthesis methodology of the functionalized nanomaterials | |
| Souri et al. | Hydrothermal fabrication of pure ZnSe nanocrystals at different microwave irradiation times and their disc-diffusion antibacterial potential against Gram negative bacteria: Bio-optical advantages | |
| CN103933066B (en) | Method for improving antibacterial activity of cephalexin through synergy of nanometer copper oxide | |
| CN114392355B (en) | Fe 3 O 4 @MoS 2 Preparation method and application of@SDS composite nanomaterial | |
| CN116395747A (en) | Manganese tetraoxide-silver sulfide Janus structure nanocomposite and preparation method thereof | |
| CN110327463A (en) | A kind of nano material and its preparation method and application comprising more Gadolinium Tungstates | |
| CN114713261A (en) | Multifunctional nano enzyme, preparation method and application thereof | |
| Nunthavarawong et al. | Antimicrobial and Antiviral Materials: Polymers, Metals, Ceramics, and Applications |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: 610000 Sichuan city of Chengdu province Wuhou District two ring road south of four No. 51 4 4 storey (Chengdu landmark building 4 layer 4) Applicant after: Chengdu Ben nano Biological Technology Co., Ltd. Address before: Seventeen Department of pharmacology, West China School of preclinical and forensic medicine, three section of Renmin South Road, Chengdu, Sichuan 610041, China Applicant before: Chengdu Ben nano Biological Technology Co., Ltd. |
|
| COR | Change of bibliographic data | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190115 Termination date: 20210630 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |